The aim of this survey was to assess the seroprevalence of antibodies against Coxiella burnetii (C. burnetii) in subjects at risk of exposure in Sicily, Southern Italy. Prevalence of IgG antibodies to C. burnetii phase II antigens was evaluated by ELISA in a group of 140 workers at risk of exposure (38 veterinarians, 38 slaughterhouse workers, 44 livestock handlers, 20 laboratory and technical personnel) included in a medical surveillance program and in 42 control subjects. Positive samples were classified as suggestive of prior exposure to C. burnetii. Antibodies against C. burnetii were detected in 88 out of 140 (62.9%) exposed workers and in 6 out of 42 (14.3%) subjects of the control group. The variables evaluated did not seem to have a significant effect on seropositivity to Coxiella with the exception of symptoms in the last 6 months preceding the survey. Our study demonstrated a high seroprevalence of C. burnetii in the group of exposed workers in comparison to non-exposed subjects of the control group. Clinical illness appears to be rare; nevertheless, physicians should consider Q fever in patients with compatible symptoms and occupational exposure to animals and their products. As aerosols represent the main route of infection in animals and humans, these workers are strongly advised to wear respiratory masks. In addition, occupational physicians should consider routine serologic evaluation and vaccination of occupationally exposed workers.